Method of encapsulating metal complex within liposomes

ABSTRACT

An object of the present invention is to provide a method of producing liposome wherein a complex of a short half-life metallic radioactive nuclide such as  99m Tc and CD is encapsulated, with radiochemical yield and purity enabling practical application. The present invention provides a method of producing a liposome wherein a complex of a short half-life metallic radioactive nuclide and ethylenedicysteine (CD) is encapsulated, which comprises mixing a complex of a short half-life metallic radioactive nuclide and N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine with an ethylenedicysteine (CD)-encapsulated liposome, and incubating the mixture.

TECHNICAL FIELD

The present invention relates to a method of producing liposomes whereina complex of a short half-life metallic radioactive nuclide such as^(99m)Tc and ethylenedicysteine (CD) is encapsulated. More specifically,the present invention relates to a method of producing liposomes whereina complex of a short half-life metallic radioactive nuclide such as^(99m)Tc is encapsulated, by using a complex of a short half-lifemetallic radioactive nuclide such as ^(99m)Tc coordinated withN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine,liposomes produced by said method, and a reagent kit for use in saidmethod.

BACKGROUND ART

The diagnostic imaging of cancer using a radioactive nuclide allows anoninvasive early diagnosis of cancer. Technetium-99m (^(99m)Tc), ametallic radioisotope (RI), is the most suitable for clinicalapplication among RIs commonly used for diagnostic imaging, since it hasa half-life (6 hours) and a γ-ray energy (141 keV) suitable fordiagnostic imaging and this nuclide is readily available as a physiologysaline solution resulted from a generator system using ⁹⁹Mo as a parentnuclide. There have been conducted many researches utilizing an antibodyor a peptide as a labeling material for the purpose of selectivelydelivering ^(99m)Tc to the tumor, and a liposome is also one of thecarrier candidates for use in diagnostic imaging. A liposome is a closedvesicle composed of a lipid bilayer membrane. A liposome draws attentionas a capsule type DDS carrier for a medicament such as chemotherapeuticdrug, a protein, a nucleic acid, etc. Since liposomes can carry a largeamount of radioactivity therein and also be formed target directed bythe adjustment of particle size and chemical modification of themembrane surface, applications of ^(99m)Tc-labeled liposomes to ahigh-sensitive diagnostic imaging of solid cancer, sentinel lymph-nodeand inflammation and infection sites are expected to be useful innuclear medicine diagnosis as well.

It has been demonstrated that since tumor tissue has increased vascularpermeability and lacks in recovery of substances via lymphatic system,macromolecules tend to infiltrate from the blood to the tumorinterstitium and accumulate therein. It is also suggested by theseproperties that liposomes permeated into the tissue interstitium are nottaken up into cells and rather stay in the cell interstitium in tumortissue. Liposomes which circulate through a blood flow, however, aremainly captured by reticuloendothelial tissues such as liver or spleen,and are removed from the blood. Although liposomes wherein anitrilotriacetic acid (NTA) complex of ⁶⁷Ga and ¹¹¹In is encapsulatedhave been well investigated as well for the purpose of diagnosticimaging of tumors and have exhibited excellent tumor accumulatingproperties in laboratory animals, they also exhibit high radioactivityaccumulation in the liver and spleen, which has been a serious obstaclefor practical application thereof. The liposomes incorporated into theliver and spleen are fused with lysosomes in the parenchymal cells, andare then metabolized. Encapsulated complexes of ⁶⁷Ga and ¹¹¹In-NTAreleased in the lysosomes at this time are water-soluble and cannotpenetrate the membrane. The complexes decompose due to their lowstability, and radioactive nuclides are accumulated in the lysosomes.This is supposed to the cause of the long-lasting radioactivityretention appearing in these tissues.

Accordingly, the present inventors considered that the non-specificretention of radioactivity would be dissipated by conferring propertiesto move from the lysosome into the blood and to be quickly excreted intothe urine on the RI complexes released after liposomes are incorporatedinto the lysosomes in the cell and metabolized and released therefrom.^(99m)Tc-ethylendicysteine (^(99m)Tc-CD) (FIG. 1) has been selected as acomplex having such properties. ^(99m)Tc-CD has two molecules of freecarboxylic acid and a stable pentavalent neutral complex structure. Itis reported that ^(99m)Tc-CD is excreted into the urine in a stablechemical form through the organic anion transporter in the kidney as inthe case of para-aminohippuric acid. According to the researches of thepresent inventors, it has been demonstrated that when ¹⁸⁶Re-CD, a CDcomplex of rhenium-186 (¹⁸⁶Re) which is a long half-life metallicradioactive nuclide of the same family as ^(99m)Tc is encapsulated inthe liposome, radioactivity accumulated in the liver and spleen can bepromptly excreted into urine as ¹⁸⁶Re-CD, and radioactivity retained inthese internal organs can be significantly reduced. These resultssuggest that the radioactivity retained in the liver or spleen can bedissipated also with the use of liposomes wherein ^(99m)Tc-CD complexesare encapsulated.

However, when a direct encapsulation approach was taken at the time ofpreparing ¹⁸⁶Re-CD, where ¹⁸⁶Re-CD complex is added to lipid as it isand then subjected to swelling, encapsulating efficiency is extremelylow as 3.2%. Accordingly, it is necessary to establish a method ofencapsulating ¹⁸⁶Re-CD or ^(99m)Tc into liposomes. Moreover, in case ofclinical use, direct encapsulation approach requires liposomes to beprepared just before use. It is practically desired, however, that theliposomes prepared beforehand are labeled when clinically used. Atechnique has been reported as an efficient and simple encapsulatingmethod for ⁶⁷Ga and ¹¹¹In, in which ⁶⁷Ga and ¹¹¹In-oxine complex havinga high lipid solubility and substitution activity is prepared and isincubated with NTA-encapsulated liposomes so that the complex penetratesthe liposome membrane and causes a ligand exchange reaction in theliposomes thereby retaining these nuclides as water-soluble chelate ⁶⁷Gaand ¹¹¹In-NTA within the liposomes (FIG. 2). When this encapsulatingmethod called ligand exchange reaction is used, the encapsulationefficiency of ⁶⁷Ga or ¹¹¹In reaches to about 90%. Another technique forobtaining ^(99m)Tc-labeled liposomes in high radiochemical yieldcomprises incubating ^(99m)Tc-hexamethyl propyleneamine oxime(^(99m)Tc-HMPAO) which is a lipophilic complex withglutathione-encapsulated liposomes, thereby reductively converting the^(99m)Tc-HMPAO within the liposome into a water-soluble decomposedproduct. The encapsulating efficiency by this technique is about 60% to90%. Recently, RI labeled liposomes used in nuclear medicine are mostcommonly prepared by this encapsulating method. This method, however,give rise to problems of radioactivity retention in the liver and spleenas mentioned above for each of the ⁶⁷Ga, ¹¹¹In and ^(99m)Tc labeledliposomes.

DISCLOSURE OF THE INVENTION

The present invention aims at solving the above-mentioned problems ofthe prior art. Namely, not only increasing the encapsulation efficiencybut also sufficiently improving the radiochemical purity of ^(99m)Tc-CDwithin the liposome is necessary for achieving a prompt excretion of theadministered radioactivity. Accordingly, an object of the presentinvention is to provide a method of producing liposome wherein a complexof a short half-life metallic radioactive nuclide such as ^(99m)Tc andCD is encapsulated, with radiochemical yield and purity enablingpractical application.

The present inventors intended to solve the above-mentioned problems andcomparatively examined two types of ^(99m)Tc complex having highmembrane permeability and substitution activity, i.e. ^(99m)Tc-HMPAO and^(99m)Tc-N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine(MRP20) (FIG. 3 by using them in the ligand exchange reaction.Furthermore, the present inventors investigated radiokinetics of theprepared ^(99m)Tc-CD encapsulated liposomes, and evaluated the utilityof the labeling method of the present invention in relation todiagnostic imaging by ^(99m)Tc as well as internal radiotherapy with¹⁸⁶Re. Consequently, it has been found that ^(99m)Tc-encapsulatedliposomes which eliminate the non-specific retention of radioactivity inthe liver and spleen can be prepared by encapsulating ^(99m)Tc-CD in theliposomes by ligand exchange reaction utilizing^(99m)Tc-N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine(^(99m)Tc-MRP20). The present invention has been completed based onthese findings.

That is, according to the present invention, there is provided a methodof producing a liposome wherein a complex of a short half-life metallicradioactive nuclide and ethylenedicysteine (CD) is encapsulated, whichcomprises mixing a complex of a short half-life metallic radioactivenuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine withan ethylenedicysteine (CD)-encapsulated liposome, and incubating themixture.

Preferably, there is provided a method of producing a^(99m)Tc-ethylenedicysteine (CD) complex-encapsulated liposome, whichcomprises mixing^(99m)Tc-N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diaminewith an ethylenedicysteine (CD)-encapsulated liposome, and incubatingthe mixture.

According to another aspect of the present invention, there is provideda liposome wherein a complex of a short half-life metallic radioactivenuclide and ethylenedicysteine (CD) is encapsulated, which is producedby mixing a complex of a short half-life metallic radioactive nuclideand N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diaminewith an ethylenedicysteine (CD)-encapsulated liposome, and incubatingthe mixture.

Preferably, there is provided a ^(99m)Tc-ethylenedicysteine (CD)complex-encapsulated liposome, which is produced by mixing^(99m)Tc-N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diaminewith an ethylenedicysteine (CD)-encapsulated liposome, and incubatingthe mixture.

According to a further another aspect of the present invention, there isprovided a diagnostic or therapeutic agent which comprises theabove-mentioned liposome.

According to a still further another aspect of the present invention,there is provided a reagent kit for use in the method of producingliposomes wherein a complex of a short half-life metallic radioactivenuclide and ethylenedicysteine (CD) is encapsulated according to thepresent invention, said kit comprising at least one substance selectedfrom the group consisting of one or more liposome forming material;ethylenedicysteine (CD); ethylenedicysteine (CD) encapsulated liposome;a short half-life metallic radioactive nuclide;N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine; and acomplex of a short half-life metallic radioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine.

Preferably, the short half-life metallic radioactive nuclide is ^(99m)Tcor its salt.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the presumed structure of ^(99m)Tc-CD. CD has nitrogen andsulfur as coordinating atoms, and forms a stable oxo-type complex with apentavalent ^(99m)Tc;

FIG. 2 shows a ⁸⁶Re-CD direct encapsulating method (A) and a ligandexchanging reaction (B) by ¹¹¹In-NTA;

-   ▪: CD, NTA-   ▴: ⁸⁶Re or ¹¹¹In-   ◯: oxine

FIG. 3 shows structures of ^(99m)Tc-HMPAO (left) and ^(99m)Tc-MRP20(right). They are ^(99m)Tc complexes having high lipid solubility andsubstitution activity, and presumably they are relatively readilyhydrolyzed and susceptible to ligand exchange reaction;

FIG. 4 shows stability of ^(99m)Tc-HMPAO and ^(99m)Tc-MRP20 in aqueoussolutions. Changes over time of the radiochemical purity of each^(99m)Tc-complex are represented assuming that the purity is 100% at the0-hour after the complexes are formed;

-   (A) ^(99m)Tc-HMPAO before purified by RP-HPLC;-   (B) ^(99m)Tc-HMPAO after purified by RP-HPLC;-   (C) ^(99m)Tc-MRP20 before purified by RP-HPLC;-   (D) ^(99m)Tc-MRP20 after purified by RP-HPLC;-   ◯: 25° C., □: 37° C.

FIG. 5 shows the effect by pH on ligand exchange reactivity of^(99m)Tc-HMPAO (A) and ^(99m)Tc-MRP20 (B), and CD. The radiochemicalabsorbance of ^(99m)Tc-CD resulted from the exchange reaction isexpressed on a vertical axis;

-   ◯: Exchange with ^(99m)Tc-complexes before purified by RP-HPLC;-   □: Exchange with ^(99m)Tc-complexes after purified by RP-HPLC:

FIG. 6 shows EP analysis results of the contents of ^(99m)Tc-CD liposome(^(99m)Tc (HMPAO)-CD liposome) prepared with ^(99m)Tc-HMPAO;

FIG. 7 shows EP analysis results (A) and RP-HPLC analysis results (B) ofthe contents of ^(99m)Tc-CD liposome (^(99m)Tc(MRP20)-CD liposome)prepared with ^(99m)Tc-MRP20;

FIG. 8 shows radiokinetics of ^(99m)Tc-labeled liposomes whenintravenously administered to mice;

-   Δ: ^(99m)Tc/GSH liposome;-   ▪: ^(99m)Tc(HMPAO)-CD liposome;-   ●: ^(99m)Tc(MRP20)-CD liposome:

FIG. 9 shows the ratio of the radioactivity excreted out of the body tothe administered radioactivity after ^(99m)Tc-CD encapsulated liposomesare administered to mice; and

-   ▪: ^(99m)Tc(HMPAO)-CD liposome administered group;-   □(hatched): ^(99m)Tc(MRP20)-CD liposome administered group;

FIG. 10 shows the analysis results by EP (A) and RP-HPLC (B) ofradioactivity which were excreted in urine after administering99mTc(MRP20)-CD liposome to mice.

BEST MODE FOR CARRYING OUT THE INVENTION

The mode for carrying out the present invention will be described below.

The method of producing a liposome wherein a complex of a shorthalf-life metallic radioactive nuclide and ethylenedicysteine (CD) isencapsulated, which is provided by the present invention, ischaracterized in that it comprises mixing a complex of a short half-lifemetallic radioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine withan ethylenedicysteine (CD)-encapsulated liposome, and incubating themixture.

Although the type of short half-life metallic radioactive nuclides whichcan be used in the present invention is not particularly limited, it ispreferably ^(99m)Tc (technetium 99m) or ^(186/188)Re, and particularlypreferably ^(99m)Tc.

The complex of a short half-life metallic radioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine(MRP20), which is used in the present invention, can be prepared bymixing an ethanol solution of MRP20 and a hydrochloric acid solution ofstannous chloride, adding a solution of ^(99m)TcO₄ ⁻ thereto and leavingthe mixture at room temperature, for example, in the case that the shorthalf-life metallic radioactive nuclide is ^(99m)Tc. When a shorthalf-life metallic radioactive nuclide other than ^(99m)Tc is used, asolution containing the corresponding short half-life metallicradioactive nuclide may be used in place of the ^(99m)TcO₄ ⁻ solution.

The ethylenedicysteine (CD) encapsulated liposomes used in the presentinvention can be prepared by any conventional method using a liposomeforming substance.

Although the liposome forming materials are not particularly limited aslong as they are normally used in the art, but phospholipids and theirderivatives and lipids other than phospholipids and their derivativesare preferably used for the purpose of providing liposomes stable in theliving body.

Examples of the above-mentioned phospholipids include natural orsynthetic phospholipids such as distearoylphosphatidylcholine,dipalmitoylphosphatidylcholine, dioleylphosphatidylcholine,phosphatidylcholine (lecithin), phosphatidylglycerol, phosphatidic acid,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, cardiolipin, soybean lecithin and egg yolk lecithin, orthose hydrogenized according to conventional methods.

Furthermore, the liposomes may be surface modified by adding a lipidderivative of a hydrophilic polymer. The lipid derivatives of ahydrophilic polymer which can be used are not particularly limited, aslong as they do not impair the structural stability of liposome.Examples thereof include polyethyleneglycol, dextran, pullulan, ficoll,polyvinyl alcohol, synthetic poly amino acids, amylose, amylopectin,mannan, cyclodextrin, pectin, carragheenan, derivatives theirof, and thelike. Particularly preferably, polyethyleneglycol and apolyethyleneglycol derivative can be used.

The liposomes used in the present invention may be used with astabilizer, antioxidant, if needed. Examples of stabilizer includesterols such as cholesterol which reduces membrane mobility; sugars suchas glycerol and sucrose, etc. Examples of antioxidant include tocopherolhomologs, for example, vitamin E, etc.

In order to prepare liposome, liposome forming material, which may be amixture of two or more types of material, dissolved in a solvent in theflask, may be evaporated under reduced pressure to remove the solvent toform a lipid thin membrane on the inner wall of the flask.

Any solvent can be used as long as it may dissolve the lipid used, andexamples thereof include halogenated hydrocarbons such as chloroform,methyl chloroform and methylene chloride, hydrocarbons such as hexaneand heptane, aromatic hydrocarbons such as benzene, toluene and xylene,ethers such as diethyl ether, diisopropyl ether, ethylene glycoldimethyl ether, tetrahydrofuran, etc.

Subsequently, after transferred to a vacuum desiccator to completelyevaporate the solvent under a reduced pressure, the lipid may be addedand swelled with a CD solution to obtain multilamellar liposomes (MLV)as a suspension. The liposomes may be further pressure filteredsubsequently through membrane filters having a pore size such as 0.2 μmor 0.05 μm to form single membrane liposomes.

The obtained liposome dispersion liquid can be separated into liposomesand substances which have not been encapsulated in the liposomes bypurification according to known methods such as gel filtration andcentrifugal separation.

To the ethylenedicysteine (CD) encapsulated liposomes as obtained above,a complex of a short half-life metallic radioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine(particularly preferably^(99m)Tc-N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine,etc.) is added, and the mixture is incubated to produce liposomeswherein a complex of a short half-life metallic radioactive nuclide andethylenedicysteine (CD) is encapsulated. The temperature and time ofincubation are not particularly limited and can be set up suitably. Forexample, liposomes wherein a complex of a short half-life metallicradioactive nuclide and ethylenedicysteine (CD) is encapsulated can beprepared by carrying out incubation for 30 minutes to several hours atroom temperature.

The liposomes wherein a complex of a short half-life metallicradioactive nuclide and ethylenedicysteine (CD) is encapsulated, whichare obtained by the above-mentioned method of the present invention, arecharacterized by the high purity of the short half-life metallicradioactive nuclide (for example, ^(99m)Tc)-CD complex. Since theradioactivity is expected to be dissipated from the liver or spleen morerapidly as the radiochemical purity of the short half-life metallicradioactive nuclide (for example, ^(99m)Tc)-CD complex inside theliposome is high, the liposomes wherein a complex of a short half-lifemetallic radioactive nuclide and ethylenedicysteine (CD) isencapsulated, which is prepared by the method of the present invention,have characteristics that the radioactivity retained in the liver orspleen is extremely decreased as compared with conventional^(99m)Tc-encapsulated liposomes and ^(99m)Tc-HMPAO-CD liposomes.Liposomes having such characteristics by themselves also fall within thescope of the present invention.

The liposomes wherein a complex of a short half-life metallicradioactive nuclide and ethylenedicysteine (CD) is encapsulated, whichare obtained by the method of the present invention, are useful as adiagnostic or therapeutic agent for various diseases including cancer ortumor.

The liposomes of the present invention can be administered to a livingbody orally or parenterally. The administration method is preferably anadministration by injection, and either one of intravenous,intramuscular, hypodermic and arterial injection, etc. can be useddepending on the location of cancer or tumor, and intravenous injectionis preferred.

When the liposomes of the present invention are administered as adiagnostic or therapeutic agent, the liposomes may be administered asthey are, but they are administered in the form of a pharmaceuticalcomposition containing the liposomes. The above-mentioned pharmaceuticalcomposition comprises the liposomes of the present invention and apharmaceutically accepted excipient and, if needed, may contain otherpharmacological agents, carriers, auxiliary agents and the like.

When the liposomes of the present invention are administered byinjection to a subject, they are preferably formed into a liquidpharmaceutical composition of a liquid.

The liquid pharmaceutical composition can be prepared as a solution orsuspension, for example, by dissolving or dispersing the liposomes ofthe present invention in a carrier such as water, a physiology salinesolution, aqueous glucose, glycerol, glycol or ethanol and, if needed,further adding an adjuvant thereto.

The pharmaceutical composition of the present invention may optionallycontain a small amount of additives such as a wetting agent, anemulsifier, a solubilizing agent, and pH buffer agent (for example,acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate,triethanolamine sodium acetate). The method of preparing such apharmaceutical composition is obvious to those skilled in the art.

Although the dosage of the liposomes of the present invention variesdepending on the purpose of administration, type and amounts of theencapsulated short half-life metallic radioactive nuclide, etc., buttypically they are administered in an amount of 0.1 mg to about 1 g foran adult patient.

Furthermore, in the present invention,^(99m)Tc-N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine(^(99m)Tc-MRP20) represented by the following formula is used:

^(99m)Tc-MRP20 can be prepared by mixing a solution ofN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine(MRP20) in ethanol and a solution of stannous chloride in hydrochloricacid, adding a solution of 99mTcO₄ ⁻ thereto and leaving the mixture atroom temperature.

Furthermore, the present invention provides a reagent kit for use in themethod of producing liposomes wherein a complex of a short half-lifemetallic radioactive nuclide and ethylenedicysteine (CD) isencapsulated, said kit comprising at least one substance selected fromthe group consisting of one or more liposome forming material;ethylenedicysteine (CD); ethylenedicysteine (CD) encapsulated liposome ;a short half-life metallic radioactive nuclide;N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine; and acomplex of a short half-life metallic radioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine.

The one or more liposome forming material and ethylenedicysteine (CD) inthe above are reagents for preparing ethylenedicysteine (CD)encapsulated liposomes, and either one of them singly or the both may becontained in the reagent kit, or alternatively ethylenedicysteine (CD)encapsulated liposomes which was prepared may be contained in thereagent kit.

Similarly, a short half-life metallic radioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine arereagents for preparing a complex of a short half-life metallicradioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine, andeither one of them singly or the both may be contained in the reagentkit, or alternatively a complex of a short half-life metallicradioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine whichwas prepared may be contained in the reagent kit.

The present invention will be described still more specifically by wayof the following examples, but the present invention is not limited bythese examples.

EXAMPLES Materials and Method for Experiment

1. General Method

A physiology saline solution eluted from ⁹⁹Mo/^(99m)Tc generator (UltraTechne Kow, Daiichi Radioisotopes Laboratory) was used as a ^(99m)TcO₄ ⁻solution. The product in the complex synthesis was confirmed by thinlayer chromatography (TLC), paper chromatography (PC), cellulose acetatemembrane electrophoresis method (EP) and reversed phase high performanceliquid chromatography (RP-HPLC). TLC was developed with a mixed solvent(4:1) of chloroform and methanol using silica gel available from Merck(Silica gel 60 F₂₅₄). PC was developed with acetonitrile 50% using afilter paper available from Whatman (No. 1). EP was conducted using acellulose acetate membrane (SELECA-V, Toyo Roshi Kaisha, Ltd.) forelectrophoresis membrane, a veronal buffer solution (pH=8.6, I=0.06,Nacalai Tesque, Inc.) for buffer solution, a fixed current (1 mA/cm) for25 minutes. RP-HPLC was conducted using COSMOSIL C₁₈-AR-300 column (4.6mm×150 mm, Nacalai Tesque, Inc.) connected with a fraction collector(Pharmacia). All the collected fractions were measured with a gammacounter (ARC-380M, Aloka). The labeled compounds were analyzed at a flowrate of 0.5 ml/min and with a mobile phase of (A) 0.01M phosphate buffersolution (pH=7.0) and acetonitrile under the condition that the ratio ofacetonitrile increased from 0 to 100% in 10 minutes, and (B) 0.0125Mphosphate buffer solution (pH=2.5) and acetonitrile under the conditionthat the ratio of acetonitrile increased from 0 to 9% in 12 minutes andincreased from 9 to 100% in 20 minutes to 40 minutes.

2. Synthesis of CD and MRP20

N,N′-Ethylenedicysteine (CD) was synthesized according to a method byBrondeau et al. (Blondeau et al. Canadian J Chem 45:49-52 and 1967).After an ammonia solution of L-thioproline (thiazolidine-4-carboxylicacid, Tokyo Kasei Kogyo Co., Ltd.) was added with metal sodium, NH₄Clwas added thereto, and the mixture was stirred overnight at roomtemperature. The generated crystals were dissolved in water andprecipitated with hydrochloric acid (yield: 23%, melting point: 251 to254° C.).

N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-one-2)-ethane-1,2-diamine(MRP20) was synthesized according to a method by Morgan et al. (Morganet al. Inorg. Chim. Acta 190:257-264, 1991). Pyrrole-2-aldehyde andethylen diamine were stirred in acetonitrile overnight, reduced withNaBH₄ in methanol solvent and extracted with chloroform under a basiccondition to obtain an intermediate compound. Subsequently, theintermediate compound and acetyl acetone was stirred in acetonitrile,extracted with chloroform and purified by column chromatography using asilica gel column (solvent ethyl acetate: methanol=5:1) andrecrystallization from benzene. Yield: 15.9%, melting point: 74 to 75°C. (document value of 75° C.).

3. Synthesis of ^(99m)Tc-HMPAO and ^(99m)Tc-MRP20

^(99m)Tc-HMPAO (^(99m)Tc-hexamethyl propyleneamine oxime) was preparedusing a kit (Cerebrotec (registered trademark), Nycomed AmershamInternational) supplied from Nihon Medi-Physics Co., Ltd. ^(99m)TcO₄ ⁻physiology saline solution was added to the kit, and it was usedimmediately after it was dissolved.

^(99m)Tc-MRP20 used was obtained by mixing 190 μl of a solution (17 mM)of MRP20 in ethanol and 10 μl of a 0.01N HCl solution (1.35 M) ofstannous chloride, and adding 200 μl of ^(99m)TcO₄ ⁻ solution theretoand leaving the mixture at room temperature for 15 minutes.

Radiochemical yields of the obtained ^(99m)Tc complexes (^(99m)Tc-HMPAO,^(99m)Tc-MRP20 and ^(99m)Tc-CD) were determined by TLC, PC, EP andRP-HPLC. Determination of radiochemical yield was conducted by cuttingthe TLC plate, the filter paper, and the cellulose acetate membraneafter dried into pieces of 5 mm width, subjecting each piece tomeasurement by gamma counter, and calculating the rate of theradioactivity at each Rf value assuming all the radioactivity of eachplate as 100%.

4. Measurement of Partition Coefficient of HMPAO and MRP20

20 μl of HMPAO or MRP20 solutions adjusted to 8 mM with a physiologysaline solution was added to 500 μl of octanol and 500 μl of a HEPESbuffer solution (pH 7.0) or Na₂HPO₄—NaOH buffer solution (pH 12.0). Themixture was allowed to stand still after stirring. Absorption of theoctanol layer and the aqueous layer was measured, and the lipophilicproperties of the two complexes were compared.

5. Stability of ^(99m)Tc Complex in an Aqueous Solution

In order to compare the stability of ^(99m)Tc-HMPAO and ^(99m)Tc-MRP20in an aqueous solution, ^(99m)Tc-HMPAO solution was diluted with 50 mMammonia buffer solution (pH 9.4) into the final ligand concentration of1 mM. The mixture was incubated in a water bath of 25° C. or 37° C., andradiochemical purity was determined by the above-mentioned analyzingmethods after predetermined time (0.5, 2, 4, 8, or 24 hours).^(99m)Tc-MRP20 aqueous solution was diluted with 50 mM phosphate buffersolution (pH 8.3), and the similar operations were effected.

For the purpose of examining the effect of free ligands on thestability, ^(99m)Tc complex solution was purified by RP-HPLC, and thepurified ^(99m)Tc complex solution eluted in 100% acetonitrile was mixedand diluted in the ratio of 1:1 with a buffer solution, and changes overtime of the radiochemical purity of the complex was examined similarlyas for the complex before purification.

6. Ligand Exchange Reactivity of ^(99m)Tc Complex with CD

CD was dissolved in 1N NaOH to 66.7 mM, diluted with a buffer solution10 times and pH adjusted with 2N HCl to obtain a CD solution. This CDsolution, ^(99m)Tc-HMPAO, and ^(99m)Tc-MRP20 solution were mixed at arate of 3:1, and after the mixture was incubated for 30 minutes at 37°C., radiochemical yield of Tc-CD was determined by EP. As a buffersolution to dilute CD, HEPES buffer solution (pH 7.0 or pH 8.3), anammonia buffer solution (pH 9.4 or pH 10.5), and a sodium phosphatebuffer solution (pH 11.2) (all 50 mM) were used. The CD concentrationafter mixing CD solution, ^(99m)Tc-HMPAO, and ^(99m)Tc-MRP20 solutionwas 5 mM, and HMPAO and MRP20 concentration was 2 mM.

7. Preparation of Liposome

After mixing and dissolving distearoyl phosphatidylcholine (DSPC, NipponOil & Fats) and cholesterol (CH, Sigma) at a ratio of 2:1 (15 μmole:7.5μmole) in 2 ml of chloroform in an eggplant type flask, the solventswere evaporated at 65° C. under reduced pressure to form a thin film oflipid on the inner wall of the flask. After transferred to a vacuumdesiccator to completely evaporate the solvent under reduced pressurefor more than four hours, an almost isotonic aqueous solution of thesubstance to be encapsulated was added to swell the lipid at 65° C.,thereby obtaining multilamellar liposomes (MLV) as a suspension. Theliposomes were pressure filtered subsequently through membrane filtershaving a pore size such as 0.2 μm and 0.05 μm (Nuclepore (registeredtrademark) and Nomura Micro Science) to form single membrane liposomes(SUV). The thus generated liposomes were subjected to gel filtrationhaving a carrier of Bio-Gel A-1.5 m (Bio-Rad) swelled with a 5% mannitolsolution (EconoColumn, 1×30 cm, Bio-Rad) and eluted with a 5% mannitolsolution for purification. Subsequently, the liposome solution wascentrifuged at 400,000 g for 20 minutes, and the precipitate wasresuspended with a physiology saline solution to prepare a liposomesolution.

8. Preparation of ^(99m)Tc-CD Encapsulated Liposome

^(99m)Tc-CD encapsulated liposomes were prepared by ligand exchangereaction. CD was dissolved in a physiology saline solution, and pH wasadjusted to 11.8 with 2N NaOH. 1.5 ml of the 5 mM CD solution was addedto a thin membrane of phospholipid produced by the above-mentionedmethod, and after liposomes were prepared, non-encapsulated CD wasremoved by gel filtration. 500 μl of the resultant purified liposomeswas diluted with the same volume of a physiology saline solution, andthen 140 μl of ^(99m)Tc-HMPAO and ^(99m)Tc-MRP20 solutions were addedthereto, and the mixture was incubated for 60 minutes at 37° C., and^(99m)Tc-CD encapsulated liposomes were obtained. The reaction solutionwas centrifuged, and the precipitated fraction was considered as afraction of ^(99m)Tc-CD encapsulated liposomes.

9. Preparation of ^(99m)Tc/GSH Liposome

^(99m)Tc/GSH liposomes were prepared according to the conventionalmethod. GSH was dissolved in 135 mM NaCl/10 mM HEPES buffer (pH 7.4),and the pH was adjusted to pH=6.7 with 2N NaOH to prepare 50 mM GSHsolution. After 1.5 ml of GSH solution was added to a thin membrane ofphospholipid to prepare liposomes, non-encapsulated GSH was removed bygel filtration. 250 μl of ^(99m)Tc-HMPAO was added to 500 μl of thepurified liposomes, and the mixture was incubated for 40 minutes at 25°C., and ^(99m)Tc/GSH liposomes were obtained. The reaction solution wascentrifuged and the precipitated fraction was considered as a fractionof ^(99m)Tc/GSH liposomes.

10. Analysis of the Contents of ^(99m)Tc-CD Encapsuled Liposome

Each of the precipitate fractions of ^(99m)Tc-CD liposome(^(99m)Tc-HMPAO-CD liposome) prepared with ^(99m)Tc-HMPAO and^(99m)Tc-CD liposome (^(99m)Tc-MRP20-CD liposome) prepared with^(99m)Tc-MRP20 was added with ethanol in such a quantity to form about250 kBq/ml solutions, and the mixtures were fully stirred and thenallowed to stand additional for 20 minutes so as to dissolve the lipidmembranes. The contents released from the liposomes were analyzed by EP.

11. Pharmacokinetics in a Normal Mouse

^(99m)Tc-CD encapsulated liposome fraction or ^(99m)Tc/GSH liposomefraction were diluted with a physiology saline solution so that theconcentration of phospholipid might become 1.4 to 1.8 μmole/ml. 0.1 mlof each liposome solution was administered to a group of five ddY malemice of five-week old from the tail vein. The mice were sacrificed bydecapitation in 10 minutes and 1, 3, 6 and 24 hours afteradministration, and each tissue was extracted and the weight andradioactivity as measured by gamma ray detection equipment ofinternal-organs were determined. The radioactivity distributed over eachinternal organs was expressed as radioactivity per 1 g of each internalorgans (% ID/g tissue) assuming the total thereof as 100%.

The radioactivity in urine was analyzed 24 hours after administration.300 μl of the collected urine was subjected to centrifugal filtrationfilter (Microcon (registered trademark), Millipore), and centrifugationwas carried out at 4° C. at 8800 rpm for 15 minutes to remove proteins,and the resultant was filtered with 0.45-μm syringe filter and analyzedby EP and RP-HPLC.

Results

1. Chemical Properties of ^(99m)Tc Compounds

^(99m)TcO₄ ⁻ migrated 8.0 cm toward the anode side in EP, Rf value inTLC developed with a mixed solvent of chloroform and methanol was 0.8 to0.9, and Rf value in PC developed with 50% acetonitrile was 0.9 to 1.0.^(99m)TcO₂ resulted by hydrolysis of reduced pentavalent ^(99m)Tc isconsidered to stay at the starting point in EP, TLC and PC. ^(99m)Tc-CDmigrated 6.0 cm toward the anode side in EP, and eluted after 22.0minutes in RP-HPLC (B). ^(99m)Tc-HMPAO did not migrate from the startingpoint in EP, and Rf value was 0.9 in TLC. Thus the radiochemical yieldwas determined by subtracting the rate of radioactivity attributable to^(99m)TcO₄ ⁻ in EP from the rate of radioactivity at about 0.9 of TLC Rfvalue. The retention time in RP-HPLC (A) was 16.9 minutes.^(99m)Tc-MRP20 which was synthesized by the method shown below did notmigrate from the starting point in EP, and exhibited Rf value of 0.8 to1.0 in PC. Thus, the radiochemical yield was calculated by determiningthe rate of radioactivity retaining at the starting point in EP and therate of radioactivity retaining at the starting point in PC. Theretention time in RP-HPLC (A) was 18.1 minutes. These results (analysisvalue of ^(99m)Tc compounds) are summarized in Table 1.

TABLE 1 Analysis value of ^(99m)Tc compounds ^(99m)TcO₄ ⁻ ^(99m)TcO₂^(99m)Tc-CD ^(99m)Tc-HMPAO ^(99m)Tc-MRP20 TLC/CH₃Cl + MeOH Rf value0.8-0.9 0 — — 0.9 PC/50% CH₃CN Rf value 0.9-1.0 0 — 0.8-1.0 — EPmigration distance (cm) 8.0 0 6.0-8.0 0 0 RP-HPLC (A) retention time(min) 4-5 — — 18.1 16.9 RP-HPLC (B) retention time (min) 4-5 — 22.0 — —2. Partition Coefficient of HMPAO and MRP20

Absorbance of HMPAO transferred to the octanol layer was below thedetection limit. The ratio of absorbances of MRP20 in the octanol layerand the water layer was 2.1±0.7 when the pH of the buffer was 7.0, and5.0±1.6 when the pH was 12.0.

3. Synthesis of ^(99m)Tc-MRP20

In the case that MRP20 was labeled with ^(99m)Tc by a conventionalprocedure, hydrolysis products of ^(99m)Tc having a negative charge weregenerated at 50% or more, and the yield of the labeled compound ofinterest was only about 35 to 40%. Accordingly, ethanol and hydrochloricacid (solvent) were purged with nitrogen for 6 hours or more, andtin/hydrochloric acid solution were added by small quantity to thereaction solution, and as result, ^(99m)Tc-MRP20 was obtained byradiochemical yield of 81 to 92%. The radiochemical purity of^(99m)Tc-MRP20 after purified by RP-HPLC (A) was almost 100%.

4. Stability of ^(99m)Tc Complex in an Aqueous Solution

Changes over time of the radiochemical purity of each complex present inthe solution were examined for the complex solutions of ^(99m)Tc-HMPAOand ^(99m)Tc-MRP20 before and after purification by RP-HPLC. The resultsare shown in FIG. 4.

Unpurified ^(99m)Tc-HMPAO was decomposed to about 40% in 30 minutesafter complex formation, and continued to be decomposed relativelymoderately. On the other hand, ^(99m)Tc-MRP20 retained a radiochemicalpurity of about 70% even after 2 hours after the complex formation andwas decomposed to about 40% at 25° C. at 4 hours after the complexformation. After purified, there were observed no significantdifferences between the stability of the two compounds at 37° C., andconversely, ^(99m)Tc-HMPAO is more stable than ^(99m)Tc-MRP20 at 25° C.After 24 hours, the radiochemical purity of ^(99m)Tc-HMPAO was 59%whereas ^(99m)Tc-MRP20 was decomposed to 6%.

5. Ligand Exchange Reactivity of ^(99m)Tc Complex with CD

Effect of pH on the ligand exchange reactivity with CD was examined forboth complexes of ^(99m)Tc-HMPAO and ^(99m)Tc-MRP20 before and afterRP-HPLC purification. The results are shown in FIG. 5.

The yield of ^(99m)Tc-CD in the exchange reaction between unpurified^(99m)Tc-HMPAO and CD increased as the pH of the mixture solutionincreased, and reached 57% at pH 11.9. Moreover, the yield or the rateof exchange reaction after purification also reached 74% at pH 11.9.

The ligand exchange reactivity between ^(99m)Tc-MRP20 and CD was muchhigher than that of ^(99m)Tc-HMPAO and CD, and exhibited ^(99m)Tc-CDyield of 90% or more at a pH within the measurement range. Particularly,purified ^(99m)Tc-MRP20 always showed 95% or more of ^(99m)Tc-CD yieldand did not affected by pH.

6. Preparation of ^(99m)Tc Encapsulated Liposome

Supernatant was sampled after ^(99m)Tc encapsulated liposomes weresubjected to centrifugal separation, and encapsulation efficiency wasdetermined by measuring the radioactivity of precipitation andsupernatant respectively. Encapsulation efficiency was determined as thevalue obtained by dividing the radioactivity of precipitation by the sumof the radioactivity of precipitation and the radioactivity ofsupernatant. The encapsulation efficiency of ^(99m)Tc-CD by the ligandexchange reaction using ^(99m)Tc-HMPAO was 66.4% while the encapsulationefficiency of ^(99m)Tc-CD using ^(99m)Tc-MRP20 was 70.0%. Theencapsulation efficiency to GSH liposome using ^(99m)Tc-HMPAO was 75.1%.

The contents of ^(99m)Tc-CD encapsulated liposomes were analyzed. Theresults are shown in FIGS. 6 and 7. As for ^(99m)Tc(HMPAO)-CD liposomesand ^(99m)Tc(MRP20)-CD liposomes, main peaks of the contents appeared inthe range from 7.5 cm to 8.0 cm toward the anode side in EP. However,the radioactivity attributable to the peak corresponding to ^(99m)Tc-CDwas only 54% of the total radioactivity inside the liposomes in case of^(99m)Tc(HMPAO)-CD liposomes. On the other hand, in case of the contentsof ^(99m)Tc(MRP20)-CD liposomes, 91.1% of radioactivity migrated 6.5 cmtoward the anode side in EP, and almost all the radioactivity was elutedat a retention time of 22.3 minutes in RP-HPLC (B).

7. Pharmacokinetics in Normal Mouse

The radiokinetics in the living body is shown in FIG. 8, when^(99m)Tc-CD encapsulated liposomes labeled with ^(99m)Tc-HMPAO or^(99m)Tc-MRP20, or ^(99m)Tc/GSH liposomes used as comparative control,was intravenously administered to a normal mouse. The radioactivitydisappeared from the blood to a similar extent in these three cases, andno significant difference was observed in the radioactivity accumulationin the liver or spleen at an early stage of administration. However,with the progress of time, increase in the radioactivity accumulation tothese organs was observed in case of ^(99m)Tc/GSH liposomes, and therearises a significant difference from the case of ^(99m)Tc-CD liposomesin 6 hours. After 24 hours, 20% of the administered radioactivity wasaccumulated per 1 g of the liver, and the accumulation tendency ofradioactivity was observed in spleen after 24 hours, and 56% of theadministered radioactivity was remained. On the other hand, in the caseof ^(99m)Tc-CD encapsulated liposomes, the radioactivity in the liverand spleen peaked out at 30 minutes and 3 hours after administrationrespectively, and decreased with time thereafter. Moreover, when twotypes of ^(99m)Tc-CD liposomes were compared, ^(99m)Tc-MRP20-CD liposomeshowed more prompt radioactivity disappearance especially in 6 hours and24 hours after administration, as compared with ^(99m)Tc(HMPAO)-CDliposome.

The amount of the radioactivity excreted out of the body in 24 hoursafter administration is shown in FIG. 9, and the results of analysis ofthe radioactivity in urine is shown in FIG. 10. The ratio of theradioactivity excreted into urine among the administrated radioactivitywas 59% for ^(99m)Tc(HMPAO)-CD liposomes administered group and 74% for^(99m)Tc(MRP20)-CD liposomes administered group. 91.2% of theradioactivity excreted in the urine after ^(99m)Tc(MRP20)-CD liposomeswere administered, migrated 6.0 cm toward the anode side in EP, and waseluted at a retention time of 22.5 minutes in RP-HPLC (B).

Consideration

Encapsulation by ^(99m)Tc-HMPAO and glutathione encapsulated liposomesmost commonly used in ^(99m)Tc labeling method of liposomes enablesencapsulation at high efficiency. However, such ^(99m)Tc encapsulatedliposomes contain reduction-decomposed substances of ^(99m)Tc-HMPAO, andnon-specific radioactivity retention caused by accumulation of ^(99m)Tccompounds in reticuloenodothelial system give rise to problems ofdegradation of the picture accuracy. On the other hand, althoughpreparation of ^(99m)Tc-CD liposomes by direct encapsulation isremarkably low in encapsulation efficiency and inferior in practicality,radiochemical purity of ^(99m)Tc-CD encapsulated in the liposomes isabout 100% theoretically, and in fact, it has been demonstrated fromprevious investigation that about 80% of the administered radioactivityis actually excreted into urine after it is administered to a mouse,exhibiting prompt radioactivity disappearance from non-target tissues.If application to diagnostic imaging is taken into consideration, it isnecessary that high radiochemical purity comparable to the directencapsulating method and high radiochemical yield comparable to theconventional labeling method are accomplished simultaneously.

When ^(99m)Tc-CD encapsulated liposomes were prepared in this exampleaccording to the ligand exchange reaction using ^(99m)Tc-HMPAO or^(99m)Tc-MRP20, the encapsulation efficiency which was 3.2% as achievedin direct encapsulating method using ¹⁸⁶Re-CD has been improved greatlyto the range from 66 to 70%. Although no significant difference inencapsulation efficiency was observed between the use of ^(99m)Tc-HMPAOand ^(99m)Tc-MRP20 as membrane permeable complexes, significantdifference has been observed in radiochemical purity of ^(99m)Tc-CDencapsulated in the liposomes after encapsulation. ^(99m)Tc-CD purity in^(99m)Tc(HMPAO)-CD liposomes is much lower as compared with^(99m)Tc-MRP20. This is attributable to the generation of theradioactive compound having a negative charge which is considered to behydrolyzed substances of ^(99m)Tc-HMPAO. The complex used as startingmaterials in a ligand exchange reaction needs to have high substitutionactivity and should be comparatively highly unstable. Therefore thegeneration reaction of a complex to be made competes with thedecomposition reaction of the complex used as the starting material.Which reaction advances in predominance strongly depends on thestability of the complex itself used as starting materials. However, notonly the stability of the complex itself but also the ligandconcentration in liposomes is presumably important. As is apparent fromthe partition coefficient of HMPAO and MRP20, free MRP20 is much morelipophilic than HMPAO. MRP20 out of the membrane easily permeatesthrough the liposome membrane by passive diffusion, thereby exhibiting ahigh concentration of free MRP20 inside the liposomes, and there is apossibility that the co-existence of resulted free ligands in excessiveamount may bring forth a state where complexes are hardly hydrolyzed.Moreover, it is shown that ^(99m)Tc-MRP20 itself has a higher stabilitythan that of ^(99m)Tc-HMPAO (FIG. 4) and that it is a complex having ahigh reactivity with CD (FIG. 5). It is considered that these factorswork in favor of a ligand exchange reaction with CD and that ^(99m)Tc-CDalso generates in high purity within liposome.

Since the prompt radioactivity disappearance from the liver or spleencan be expected as the radiochemical purity of ^(99m)Tc-CD insideliposomes is high. Accordingly the reason why ^(99m)Tc-MRP20-CD liposomehas reduced the radioactivity retention in the liver or spleen moresignificantly than conventional ^(99m)Tc encapsulated liposomes and^(99m)Tc-HMPAO-CD liposomes is supposed to be the result that the purityof inner ^(99m)Tc-CD gave a significant effect on the distribution ofthe radioactivity in the body after administration. This is alsosupported by the fact that 74% of the administered radioactivity wasexcreted into the urine and 91% thereof was detected with the chemicalform of ^(99m)Tc-CD. These results have revealed that ligand exchangingreaction using ^(99m)Tc-MRP20 enables the production of highradiochemical yield of ^(99m)Tc-CD encapsulated liposomes which reducesnon-specific radioactivity retention involved in conventional method.The encapsulating method of the present invention is useful to produce^(99m)Tc labeled liposomes which enables high precision diagnosticimaging. Moreover, it is expected that the encapsulating method of thepresent invention gives fundamental knowledge for the production of cellkilling ^(186/188)Re labeled liposome for the purpose of innerradiotherapy for cancers.

INDUSTRIAL APPLICABILITY

Since ^(99m)Tc-MRP20 which is a membrane permeable complex can give^(99m)Tc-CD in high yield through a ligand exchange reaction with CD,the ligand exchange reaction using ^(99m)Tc-MRP20 according to thepresent invention enables production of ^(99m)Tc-CD encapsulatedliposomes at high radiochemical yield and high purity. Moreover, it hasbeen demonstrated that ^(99m)Tc-CD encapsulated liposomes produced bythe present method greatly reduces the non-specific radioactivityretention in the liver and spleen involved in conventional method. Thus,the present invention enables to improve diagnostic imaging accuracyusing ^(99m)Tc labeled liposomes. Moreover, the method of the presentinvention is also applicable to the production of cell-killing^(186/188)Re labeled liposomes for the purpose of inner radiotherapy ofcancers.

1. A method of producing a liposome wherein a complex of a shorthalf-life metallic radioactive nuclide and ethylenedicysteine (CD) isencapsulated, which comprises mixing a complex of a short half-lifemetallic radioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine withan ethylenedicysteine (CD)-encapsulated liposome, and incubating themixture.
 2. A method of producing a ^(99m)Tc-ethylenedicysteine (CD)complex-encapsulated liposome, which comprises mixing^(99m)Tc-N-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diaminewith an ethylenedicysteine (CD)-encapsulated liposome, and incubatingthe mixture.
 3. A reagent kit for use in the method of producingliposomes wherein a complex of a short half-life metallic radioactivenuclide and ethylenedicystein (CD) is encapsulated according to claim 1,said kit comprising ethylenedicysteine (CD) encapsulated liposome and acomplex of a short half-life metallic radioactive nuclide andN-[2-(1H-pyrrolylmethyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine. 4.The reagent kit of claim 3 wherein the short half-life metallicradioactive nuclide is ^(99m)Tc or its salt.